Purinergic signaling inhibits human acute myeloblastic leukemia cell proliferation, migration, and engraftment in immunodeficient mice.

Extracellular ATP and UTP nucleotides increase the proliferation and engraftment potential of normal human hematopoietic stem cells via the engagement of purinergic receptors (P2Rs). In the present study, we show that ATP and UTP have strikingly opposite effects on human acute myeloblastic leukemia (AML) cells. Leukemic cells express P2Rs. ATP-stimulated leukemic cells, but not normal CD34+ cells, undergo down-regulation of genes involved in cell proliferation and migration, whereas cell-cycle inhibitors are up-regulated. Functionally, ATP induced the inhibition of proliferation and accumulation of AML cells, but not of normal cells, in the G0 phase of the cell cycle. Exposure to ATP or UTP inhibited AML-cell migration in vitro. In vivo, xenotransplantation experiments demonstrated that the homing and engraftment capacity of AML blasts and CD34+CD38- cells to immunodeficient mice BM was significantly inhibited by pretreatment with nucleotides. P2R-expression analysis and pharmacologic profiling suggested that the inhibition of proliferation by ATP was mediated by the down-regulation of the P2X7R, which is up-regulated on untreated blasts, whereas the inhibition of chemotaxis was mainly mediated via P2Y2R and P2Y4R subtypes. We conclude that, unlike normal cells, P2R signaling inhibits leukemic cells and therefore its pharmacologic modulation may represent a novel therapeutic strategy.

FGF-2 expands murine hematopoietic stem and progenitor cells via proliferation of stromal cells, c-Kit activation, and CXCL12 down-regulation.

Cytokine-induced expansion of hematopoietic stem and progenitor cells (HSPCs) is not fully understood. In the present study, we show that whereas steady-state hematopoiesis is normal in basic fibroblast growth factor (FGF-2)-knockout mice, parathyroid hormone stimulation and myeloablative treatments failed to induce normal HSPC proliferation and recovery. In vivo FGF-2 treatment expanded stromal cells, including perivascular Nestin(+) supportive stromal cells, which may facilitate HSPC expansion by increasing SCF and reducing CXCL12 via mir-31 up-regulation. FGF-2 predominantly expanded a heterogeneous population of undifferentiated HSPCs, preserving and increasing durable short- and long-term repopulation potential. Mechanistically, these effects were mediated by c-Kit receptor activation, STAT5 phosphorylation, and reduction of reactive oxygen species levels. Mice harboring defective c-Kit signaling exhibited abrogated HSPC expansion in response to FGF-2 treatment, which was accompanied by elevated reactive oxygen species levels. The results of the present study reveal a novel mechanism underlying FGF-2-mediated in vivo expansion of both HSPCs and their supportive stromal cells, which may be used to improve stem cell engraftment after clinical transplantation.

Enhanced c-Met activity promotes G-CSF-induced mobilization of hematopoietic progenitor cells via ROS signaling.

Mechanisms governing stress-induced hematopoietic progenitor cell mobilization are not fully deciphered. We report that during granulocyte colony-stimulating factor-induced mobilization c-Met expression and signaling are up-regulated on immature bone marrow progenitors. Interestingly, stromal cell-derived factor 1/CXC chemokine receptor-4 signaling induced hepatocyte growth factor production and c-Met activation. We found that c-Met inhibition reduced mobilization of both immature progenitors and the more primitive Sca-1(+)/c-Kit(+)/Lin(-) cells and interfered with their enhanced chemotactic migration to stromal cell-derived factor 1. c-Met activation resulted in cellular accumulation of reactive oxygen species by mammalian target of rapamycin inhibition of Forkhead Box, subclass O3a. Blockage of mammalian target of rapamycin inhibition or reactive oxygen species signaling impaired c-Met-mediated mobilization. Our data show dynamic c-Met expression and function in the bone marrow and show that enhanced c-Met signaling is crucial to facilitate stress-induced mobilization of progenitor cells as part of host defense and repair mechanisms.

Inhibition of erythropoiesis in malaria anemia: role of hemozoin and hemozoin-generated 4-hydroxynonenal.

Severe malaria anemia is characterized by inhibited/altered erythropoiesis and presence of hemozoin-(HZ)-laden bone-marrow macrophages. HZ mediates peroxidation of unsaturated fatty acids and production of bioactive aldehydes such as 4-hydroxynonenal (HNE). HZ-laden human monocytes inhibited growth of cocultivated human erythroid cells and produced HNE that diffused to adjacent cells generating HNE-protein adducts. Cocultivation with HZ or treatment with low micromolar HNE inhibited growth of erythroid cells interfering with cell cycle without apoptosis. After HZ/HNE treatment, 2 critical proteins in cell-cycle regulation, p53 and p21, were increased and the retinoblastoma protein, central regulator of G1-to-S-phase transition, was consequently hypophosphorylated, while GATA-1, master transcription factor in erythropoiesis was reduced. The resultant decreased expression of cyclin A and D2 retarded cell-cycle progression in erythroid cells and the K562 cell line. As a second major effect, HZ and HNE inhibited protein expression of crucial receptors (R): transferrinR1, stem cell factorR, interleukin-3R, and erythropoietinR. The reduced receptor expression and the impaired cell-cycle activity decreased the production of cells expressing glycophorin-A and hemoglobin. Present data confirm the inhibitory role of HZ, identify HNE as one HZ-generated inhibitory molecule and describe molecular targets of HNE in erythroid progenitors possibly involved in erythropoiesis inhibition in malaria anemia.

Efficient transcriptional targeting of human hematopoietic stem cells and blood cell lineages by lentiviral vectors containing the regulatory element of the Wiskott-Aldrich syndrome gene.

The ability to effectively transduce human hematopoietic stem cells (HSCs) and to ensure adequate but "physiological" levels of transgene expression in different hematopoietic lineages represents some primary features of a gene-transfer vector. The ability to carry, integrate, and efficiently sustain transgene expression in HSCs strongly depends on the vector. We have constructed lentiviral vectors (LV) containing fragments of different lengths of the hematopoietic-specific regulatory element of the Wiskott-Aldrich syndrome (WAS) gene-spanning approximately 1,600 and 170 bp-that direct enhanced green fluorescent protein (EGFP) expression. The performance of vectors carrying the 1,600 and 170 bp fragments of the WAS gene promoter was compared with that of a vector carrying the UbiquitinC promoter in human cord blood CD34(+) cells and their differentiated progeny both in vitro and in vivo in non-obese diabetic mice with severe combined immunodeficiency. All vectors displayed a similar transduction efficiency in CD34(+) cells and promoted long-term EGFP expression in different hematopoietic lineages, with an efficiency comparable to, and in some instances (for example, the 170-bp promoter) superior to, that of the UbiquitinC promoter. Our results clearly demonstrate that LV containing fragments of the WAS gene promoter/enhancer region can promote long-term transgene expression in different hematopoietic lineages in vitro and in vivo and represent suitable and highly efficient vectors for gene transfer in gene-therapy applications for different hematological diseases and for research purposes. In particular, the 170-bp carrying vector, for its reduced size, could significantly improve the transduction/expression of large-size genes.

Sustained long-term engraftment and transgene expression of peripheral blood CD34+ cells transduced with third-generation lentiviral vectors.

As mobilized peripheral blood (MPB) represents an attractive cell source for gene therapy, we investigated the ability of third-generation lentiviral vectors (LVs) to transfer the enhanced green fluorescent protein gene into MPB CD34(+) cells in culture conditions allowing expansion of transplantable human hematopoietic stem cells. To date, few studies have reported transduction of MPB cells with vesicular stomatitis virus G pseudotyped LVs. The critical issue remains whether primitive, hematopoietic repopulating cells have, indeed, been transduced. In vitro (5 weeks' culture in FLT3 ligand + thrombopoietin + stem cell factor + interleukin 6) and in vivo (serial transplantation in NOD/SCID mice) experiments show that MPB CD34(+) cells can be effectively long-term transduced by LV and maintain their proliferation, self-renewal, and multilineage differentiation potentials. We show that expansion following transduction improves the engraftment of transduced MPB CD34(+) (4.6-fold expansion of SCID repopulating cells by limiting dilution studies). We propose ex vivo expansion after transduction as an effective tool to improve gene therapy protocols with MPB. Disclosure of potential conflicts of interest is found at the end of this article.

Blood-forming stem cells are nervous: direct and indirect regulation of immature human CD34+ cells by the nervous system.

The nervous system regulates immunity through hormonal and neuronal routes as part of host defense and repair mechanism. Here, we review the emerging evidence for regulation of human hematopoietic stem and progenitor cells (HSPC) by the nervous system both directly and indirectly via their bone marrow (BM) niche-supporting stromal cells. Functional expression of several neurotransmitter receptors was demonstrated on HSPC, mainly on the more primitive CD34(+)/CD38(-/low) fraction. The myeloid cytokines, G-CSF and GM-CSF, dynamically upregulate neuronal receptor expression on human HSPC. This is followed by an increased response to neurotransmitters, leading to enhanced proliferation and motility of human CD34(+) progenitors, repopulation of the murine BM and their egress to the circulation. Importantly, recent observations showed rapid mobilization of human HSPC to high SDF-1 expressing ischemic tissues of stroke individuals followed by neoangiogenesis, neurological and functional recovery. Along with decreased levels of circulating immature CD34(+) cells and SDF-1 blood levels found in patients with early-stage Alzheimer's disease, these findings suggest a possible involvement of human HSPC in brain homeostasis and thus their potential clinical applications in neuropathology.

Refreezing of cord blood hematopoietic stem cells for allogenic transplantation: in vitro and in vivo validation of a clinical phase I/II protocol in European and Italian Good Manufacturing Practice conditions.

OBJECTIVE: Several requirements need to be fulfilled for clinical use of expanded hematopoietic stem cells (HSCs). Because most cord blood (CB) samples are frozen in single bags and only an aliquot ( approximately 25%) of the blood can be expanded, the thawing and refreezing of samples must be validated in the current European and Italian Good Manufacturing Practice (eIGMP) conditions. Here, we describe in vitro and in vivo validation of the phase I/II protocol for CD34+ expansion of thawed CB units according to the current Cell Therapy Products (CTPs) Guidelines. MATERIALS AND METHODS: CB units were thawed and 25% of the total volume was processed for CD34+ selection by CliniMACS. The 75% of the unit was immediately refrozen. CD34+ cells were expanded for 3 weeks with stem cell factor, Flt-3/Flk-2 ligand, thrombopoietin, and interleukin-6. RESULTS: In vitro results demonstrated that this culture system induces expansion of thawed CD34+ (median value = 8.3). In vivo data demonstrated that after culture, the final CTPs maintain their repopulating ability in nonobese diabetic severe combined immunodeficient (SCID) mice. Limiting dilution assays performed by injecting decreasing doses of expanded CD34+ cells revealed that the frequency of SCID repopulating cells after ex vivo expansion is 1:8,034. Analyses for sterility, viability, cell senescence, and cytogenetic assessment demonstrated that expansion procedures in eIGMP conditions are safe for clinical protocols. CONCLUSIONS: This offers promising new options for expansion of allogenic HSCs and also for autologous usage in transplantation and other cell therapy protocols.

Somatic mutations of epidermal growth factor receptor in bile duct and gallbladder carcinoma.

OBJECTIVE: Conventional therapies are still unsuccessful in patients with carcinoma arising from the biliary tract. Somatic mutations of the epidermal growth factor receptor (EGFR) gene and the activation of its downstream pathways predict the sensitivity to small-molecule inhibitors in non-small cell lung carcinoma. Therefore, we analyzed EGFR mutations and related pathways in gallbladder and bile duct carcinomas to consider the possible application of these alternative therapeutic strategies. EXPERIMENTAL DESIGN: Forty paraffin-embedded samples, including intrahepatic or extrahepatic cholangiocarcinoma and gallbladder carcinoma, were studied after tumor cell isolation by laser microdissection and sequencing of EGFR tyrosine kinase domain (exons 18-21). Activation of EGFR pathway was studied by evaluating phosphorylation of mitogen-activated protein kinase and Akt. RESULTS: None of the 40 specimens had mutations in exon 18; one had one missense point mutation in exon 19, two in exon 20, and three in exon 21. In addition, 36 of 40 specimens had the same silent mutation at codon 787 in exon 20, which was also found in peripheral blood cells from healthy donors. Tumor samples harboring EGFR mutation had phosphorylation of one or both downstream transducers analyzed. CONCLUSIONS: This is the first evidence of somatic mutations of the EGFR gene in bile duct carcinoma. Our findings suggest that a subgroup of patients with cholangiocarcinoma or gallbladder carcinoma exhibits somatic mutations of EGFR in the tyrosine kinase domain that can elicit cell signals sustaining survival and proliferation. These tumors might be further evaluated for their susceptibility to small-molecule inhibitor treatment.

Multilineage engraftment of refrozen cord blood hematopoietic progenitors in NOD/SCID mice.

Seven cord blood (CB) units were tested for their capacity to repopulate irradiated NOD/SCID mice after one or two successive cryopreservation procedures. In primary transplants with frozen or refrozen CB cells we observed equivalent human colonies and percentages of human CD45+ cells, with multilineage engraftment. In secondary transplants flow cytometry and polymerase chain reaction for the a satellite region of chromosome 17 showed equivalent levels of human engraftment. Since CB units have, to date, mainly been stored in individual bags, our results suggest new options for optimizing the timing of infusions of expanded and non-expanded progenitors in transplants.

Involvement of chemokine receptor 4/stromal cell-derived factor 1 system during osteosarcoma tumor progression.

Despite intensive chemotherapy and surgery treatment, lung and bone metastasis develop in about 30% of patients with osteosarcoma. Mechanisms for this preferential metastatic behavior are largely unknown. We investigated the role of the chemokine receptor 4 (CXCR4)/stromal cell-derived factor 1 (SDF-1) system to drive the homing of osteosarcoma cells. We analyzed the expression of the CXCR4 and SDF-1 proteins on several osteosarcoma cell lines and the effects of SDF-1 on migration, adhesion, and proliferation of these cancer cells. In vitro assays showed that the migration of osteosarcoma cells expressing CXCR4 receptor follows an SDF-1 gradient and that their adhesion to endothelial and bone marrow stromal cells is promoted by SDF-1 treatment. Moreover, the production of matrix metalloproteinase-9 is increased after SDF-1 exposure. We finally proved in a mouse model our hypothesis of the CXCR4/SDF-1 axis involvement in the metastatic process of osteosarcoma cells. Development of lung metastasis after injection of osteosarcoma cells was prevented by the administration of a CXCR4 inhibitor, the T134 peptide. These data show a possible explanation for the preferential osteosarcoma metastatic development into the lung, where SDF-1 concentration is high, and suggest that molecular strategies aimed at inhibiting the CXCR4/SDF-1 pathway, such as small-molecule inhibitors or anti-CXCR4 antibodies, might prevent the dissemination of osteosarcoma cells.

Feasibility of cord blood stem cell manipulation with high-energy shock waves: an in vitro and in vivo study.

OBJECTIVE: Cord blood CD34+ cells are more uncommitted than their adult counterparts as they can be more easily maintained and expanded in vitro and transduced with lentiviral vectors. The aim of this study was to evaluate whether pretreatment with high-energy shock waves (HESW) could further enhance the expansion of cord blood progenitors and the transduction efficiency with lentiviral vectors. METHODS: Human cord blood CD34+ cells underwent HESW treatment with a wide range of energy and number of shots (from 0.22 mJ/mm2 to 0.43 mJ/mm2 and from 200 to 1500 shots). Cells were then evaluated both for their in vitro expansion ability and in vivo engraftment in primary, secondary, and tertiary NOD/SCID mice. The transduction efficiency with a lentiviral vector (LV) was also evaluated in vitro and in vivo. RESULTS: Cell viability following HESW ranged from 75 to 92%. Pretreatment with HESW significantly improved early progenitor cell expansion after short-term suspension culture. Upon transplantation in primary NOD/SCID mice, the HESW treatment enhanced progenitor cell engraftment (total human CD45(+)CD34+ cells were 10% in controls and 14.5% following HESW, human CD45(+)CD34(+)CD38(-) cells were 0.87% in controls and 1.8% following HESW). HESW treatment enhanced the transduction of a GFP+ lentiviral vector (e.g., at day 42 of culture 6.5% GFP+ cells in LV-treated cell cultures compared to 11.4% of GFP+ cells in HESW-treated cell cultures). The percentage of human GFP+ cell engrafting NOD/SCID mice was similar (34% vs 26.4% in controls); however, the total number of human cells engrafted after HESW was higher (39.6% vs 15%). CONCLUSION: The pretreatment of CD34+ cells with HESW represents a new method to manipulate the CD34+ population without interfering with their ability to both expand and engraft and it might be considered as a tool for genetic approaches.

Vasculogenic potential of long term repopulating cord blood progenitors.

In the adult, involvement of bone marrow-derived circulating endothelial progenitor cells (EPCs) in tissue revascularization (vasculogenesis) and the cooperation of hematopoietic cell subsets in supporting this process have been described in different experimental animal models. However, the effective contribution of such cells in restoring organ vascularization in a clinical setting needs to be clarified. In this study, a mouse transplantation model was engrafted by human cord blood hematopoietic stem and progenitor cells to follow the behavior of donor-derived endothelial and hematopoietic cells in the presence of a localized source of an angiogenic inducer. Human endothelial markers (CD31+/CD45-, VE-cadherin+) were always detectable in the bone marrow of transplanted mice, while they were only randomly detectable in peripheral neovascularization sites. To investigate the ability of human transplanted hematopoietic stem cells to support new vessel formation in response to altered homeostatic conditions, chimeric mice were further treated by systemic injection of human mononuclear cells (MNCs). Our data indicate that MNC administration in transplanted mice enhances vasculogenesis in the newly formed vessels. Taken together these results suggest that human-derived EPCs, long-term engrafting a xenotransplantation model, have hematopoietic and endothelial developmental potential, which can be modulated by altering the physiological conditions of host microenvironment.

Expression of the c-ErbB-2/HER2 proto-oncogene in normal hematopoietic cells.

The HER2/c-ErbB-2 proto-oncogene is overexpressed in 25-30% of human breast cancers. We previously reported the c-ErbB-2 transcript in mononuclear cells (MNC) from bone marrow (BM), peripheral blood (PB), and mobilized PB (MPB). Here, we describe extensively the expression pattern of c-ErbB-2 mRNA and protein in normal adult hematopoietic tissue and cord blood (CB)-derived cells. Quantitative reverse transcriptase-polymerase chain reaction shows that the c-ErbB-2 transcript is expressed in hematopoietic cells at low levels if compared with normal epithelial and breast cancer cells. The c-ErbB-2 protein was detected predominantly in MNC from PB and CB by Western blot analysis. Flow cytometry revealed that CD15+, CD14+, and glycophorin A+ subpopulations express c-ErbB-2 protein, whereas lymphocytes are c-ErbB-2-negative. The c-ErbB-2 expression is higher in CB MNC. More than 90% of BM- and MPB-derived CD34+ progenitors are c-ErbB-2-negative; by contrast, 5-40% of CB-derived CD34+ progenitors express c-ErbB-2. We found that c-ErbB-2 protein is up-regulated during cell-cycle recruitment of progenitor cells. Similarly, it increases in mature, hematopoietic proliferating cells. This study reports the first evidence that the c-ErbB-2 receptor is correlated to the proliferating state of hematopoietic cells. Studies in progress aim to clarify the role of c-ErbB-2 in regulation of this process in hematopoietic tissues.

Proliferative senescence in hematopoietic stem cells during ex-vivo expansion.

The good outcome of hematopoietic stem cell (HSC) transplantation is hampered by low doses of CD34+ cell infusion. Transplanted HSCs undergo a replicative stress that causes accelerated senescence due to rapid telomere shortening. The expansion of human cord blood HSCs is instrumental in obtaining a large number of "good quality" cells, in terms of telomere length and telomerase activity compared to adult HSCs.

Ex vivo expansion of human adult stem cells capable of primary and secondary hemopoietic reconstitution.

OBJECTIVE: Ex vivo expansion of human hemopoietic stem cells (HSC) is an important issue in transplantation and gene therapy. Encouraging results have been obtained with cord blood, where extensive amplification of primitive progenitors was observed. So far, this goal has been elusive with adult cells, in which amplification of committed and mature cells, but not of long-term repopulating cells, has been described. METHODS: Adult normal bone marrow (BM) and mobilized peripheral blood (MPB) CD34(+) cells were cultured in a stroma-free liquid culture in the presence of Flt-3 ligand (FL), thrombopoietin (TPO), stem cell factor (SCF), interleukin-6 (IL-6), or interleukin-3 (IL-3). Suitable aliquots of cells were used to monitor cell production, clonogenic activity, LTC-IC output, and in vivo repopulating capacity. RESULTS: Here we report that BM and MPB HSC can be cultured in the presence of FL, TPO, SCF, and IL-6 for up to 10 weeks, during which time they proliferate and produce large numbers of committed progenitors (up to 3000-fold). Primitive NOD/SCID mouse repopulating stem cells (SRC) are expanded sixfold after 3 weeks (by limiting dilution studies) and retain the ability to repopulate secondary NOD/SCID mice after serial transplants. Substitution of IL-6 with IL-3 leads to a similarly high production of committed and differentiated cells but only to a transient (1 week) expansion of SRC(s), which do not possess secondary repopulation capacity. CONCLUSION: We report evidence to show that under appropriate culture conditions, adult human SRC can also be induced to expand with limited differentiation.

Hemozoin- and 4-hydroxynonenal-mediated inhibition of erythropoiesis. Possible role in malarial dyserythropoiesis and anemia.

Malarial anemia involves destruction of parasitized and non-parasitized red blood cells and dyserythropoiesis. Malarial pigment, hemozoin (HZ), is possibly implicated in dyserythropoiesis. We show that supernatants of HZ and HZ-fed-monocytes, and 4-hydroxynonenal generated by them, inhibited progenitor growth.

MEK-ERK pathway is expressed but not activated in high proliferating, self-renewing cord blood hematopoietic progenitors.

INTRODUCTION: The expression, activity and functions of mitogen-activated protein (MAP) kinases in primary human hematopoietic progenitors (HP) have not yet been fully clarified. MATERIAL AND METHODS: To perform our experiments we used a stroma-free cell culture system in which the combination of FLT3 ligand (FL), stem cell factor (SCF) and thrombopoietin (TPO) induces massive expansion and proliferation of cord blood HP. The addition of IL-3 results in a rapid decrease of HP due to the prevalence of maturation and cell death. To detect extracellular regulated kinase (ERK) immunoenzymatic activity we recovered HP from FL, SCF and TPO stimulated long term cultures (LTC) after four weeks of culture. Some samples were recovered 16 h after addition of IL-3 to the LTC. We selectively immunoprecipitated p44/42 ERK kinase from 245 microg of cell lysates. We then analysed dual-phosphorylation of ERK-activating kinase-kinase (p45 MEK1/2) and of p44 ERK1 and p42 ERK2, and investigated MEK and ERK expression. RESULTS: ERK activity, MEK1, and p42 and p44 ERK dual-phosphorylation were undetectable in the expanding, greatly proliferating and self-renewing HP. However, after addition of IL-3 sustained (still detectable 16 h after the stimulus) and high levels of ERK activity and dual-phosphorylation of the kinases were seen. The levels of MEK and ERK expression were stable in the different phases. CONCLUSIONS: These findings add new information on the intracellular mechanisms of HP and help explain the very low levels of hematopoietic toxicity recently seen when treating cancer with down-modulators of ERK activity.

Cytokine-induced killer cells eradicate bone and soft-tissue sarcomas.

Unresectable metastatic bone sarcoma and soft-tissue sarcomas (STS) are incurable due to the inability to eradicate chemoresistant cancer stem-like cells (sCSC) that are likely responsible for relapses and drug resistance. In this study, we investigated the preclinical activity of patient-derived cytokine-induced killer (CIK) cells against autologous bone sarcoma and STS, including against putative sCSCs. Tumor killing was evaluated both in vitro and within an immunodeficient mouse model of autologous sarcoma. To identify putative sCSCs, autologous bone sarcoma and STS cells were engineered with a CSC detector vector encoding eGFP under the control of the human promoter for OCT4, a stem cell gene activated in putative sCSCs. Using CIK cells expanded from 21 patients, we found that CIK cells efficiently killed allogeneic and autologous sarcoma cells in vitro. Intravenous infusion of CIK cells delayed autologous tumor growth in immunodeficient mice. Further in vivo analyses established that CIK cells could infiltrate tumors and that tumor growth inhibition occurred without an enrichment of sCSCs relative to control-treated animals. These results provide preclinical proof-of-concept for an effective strategy to attack autologous sarcomas, including putative sCSCs, supporting the clinical development of CIK cells as a novel class of immunotherapy for use in settings of untreatable metastatic disease.

Cytokine-induced killer (CIK) cells as feasible and effective adoptive immunotherapy for the treatment of solid tumors.

INTRODUCTION: Cytokine-induced killer (CIK) cells are heterogeneous ex vivo-expanded T lymphocytes with mixed T-NK phenotype and endowed with a wide MHC-unrestricted antitumor activity. CIK cells can be expanded from peripheral blood mononuclear cells (PBMC) cultured with the timed addition of IFN-γ, Ab anti-CD3 and IL2. A consistent subset of mature CIK cells presents a CD3(+)CD56(+) phenotype. The CD3(+)CD56(+) cellular subset is the main responsible for the tumor-killing activity, mostly mediated by the interaction of NKG2D receptor with MHC-unrestricted ligands (MIC A/B; ULBPs) on tumor cells. AREAS COVERED: In the present work, we described the biologic characteristics of CIK cells, focusing on those aspects that may favor their clinical translation. We reviewed preclinical data and analyzed reports from clinical trials. A specific paragraph is dedicated to future research perspectives in the field. EXPERT OPINION: CIK cells represent a realistic new option in the field of cancer immunotherapy. Crucial issues, favoring their clinical translation, are the easy availability of large amounts of expanded CIK cells and their MHC-unrestricted tumor killing, potentially effective against many tumor types. Intriguing future perspectives and open challenges are the investigation of synergisms with other immunotherapy approaches, targeted therapies or even conventional chemotherapy.